This project proposes to characterize the structure and activities of an endogenous growth inhibitor that has been purified from medium "conditioned" by exposure to density-inhibited mouse 3T3 fibroblasts. This factor (Mr 13,000) is designated FGR-s (13 K), which stands for Fibroblast Growth Regulator that is shed or secreted into the medium in a soluble form. We have generated a monoclonal antibody that binds this Mr = 13,000 polypeptide and that neutralizes its growth inhibitory activity. Partial amino acid sequence results indicate that FGR-s (13 K) is homologous to a family of thiol proteinase inhibitors called the Cystatins. The specific objectives of the proposed research are: (1) to identify a cDNA for FGR-s (13 K) and to determine the nucleotide sequence of the coding region in the cDNA; (2) to express the recombinant FGR-s (13 K) polypeptide in E. coli and to purify the active protein form the enriched source; (3) to analyze the components of the target cell with which FGR- s (13 K) interacts; (4) to analyze the production and binding of FGR-s (13 K) in normal versus transformed cells, fibroblasts versus other cell types, cells at different densities and at different densities and at different stages of the cell cycle; (5) to probe the cellular sites of action of FGR-s (13 K) and to study the biochemcial mechanism of growth control. The successful attainment of the initial goals would provide a good source of FGR-s (13 K) for the analysis of its mechanism of action. These latter studies should, in turn, contribute to our understanding of the general biological significance of endogenous growth inhibitors in cellular homeostasis.